1. Field of the Invention
The present invention relates to the discovery of the nucleic acid sequence and amino acid sequence encoding a newly identified G. vaginalis pore-forming toxin called vaginolysin (VLY), and to methods for diagnosing, preventing and treating G. vaginalis and bacterial vaginosis (BV). Other aspects are related to vaccines that include a toxoid form of VLY as an immunogen.
2. Description of the Related Art
Bacterial vaginosis (BV) is the most common vaginal infection worldwide and is associated with significant adverse consequences including and preterm labor and delivery (40, 41), post-partum endometritis, (42) and an increased risk of HIV acquisition. (43-45) Reported prevalence rates range from 10-40% depending upon the population studied. (46) However, suboptimal methods of diagnosis and a high percentage of asymptomatic patients make the true prevalence of BV difficult to ascertain. Gardnerella vaginalis is a bacterial species associated with bacterial vaginosis (BV).
The pathogenesis of BV remains poorly understood. It is most commonly defined as a pathological state characterized by the loss of normal vaginal flora, particularly Lactobacillus species, and overgrowth of other microbes including Gardnerella vaginalis, Bactericides species, Mobiluncus species, and Mycoplasma hominis. Recent data however, suggest a primary role for G. vaginalis as a specific and sexually transmitted etiological agent in BV, as was initially postulated by Gardner and Dukes in 1955. (47-49)
Alterations of both local host immunity and the genital tract microflora appear to contribute to the pathogenesis of BV (39), which can be difficult to eradicate even using targeted antimicrobial therapy (4). In addition, randomized trials of antibiotics for the prevention of BV-associated preterm birth have not shown consistently beneficial effects, suggesting that host inflammatory responses set in motion early in the course of disease may contribute significantly to the consequences of infection (26, 27).
In the 1950s, Leopold (25) and then Gardner and Dukes (14) observed abundant small, pleomorphic gram-variable rods in the genital tract of women with BV. This organism, first called Haemophilus vaginalis (13) and repeatedly renamed as more information about its characteristics became available (reviewed in (5)), is now classified as Gardnerella vaginalis, the sole member of the genus Gardnerella (16, 30). Phylogenetic analysis based on 16S rRNA places Gardnerella in the gram-positive family Bifidobacteriales. An abundance of G. vaginalis and a paucity of Lactobacillus species are characteristic of a BV-associated microflora, but the relative contribution of G. vaginalis to the pathogenesis of BV is not clear. G. vaginalis is present in essentially all cases of BV but can also be detected in a minority of asymptomatic women (1). Likewise, several groups have demonstrated that the vaginal microflora is exceedingly complex in BV where the vaginal mucosa is host to many non-Gardnerella organisms (12, 18, 20). Mechanistic studies of BV and its adverse consequences have been limited by the absence of definitive diagnostic testing and a suitable animal model (22, 23, 26). Existing methods of diagnosis for BV are suboptimal and frequently underutilized by practitioners. A recent study by Hogan et al. reports that the prevalence of BV among pregnant women varies greatly depending on the diagnostic criteria used. (51) Established in 1983, Amsel's criteria are widely accepted as the best available means for diagnosing BV in the clinical setting, however, these criteria are complex, somewhat subjective, and necessitate that microscopy equipment be present on site. (52, 53) The Nugent scoring system for interpretation of Gram-stained vaginal smears was put forth in an attempt to standardize diagnosis of BV and increase inter-rater reliability. (54) While the Nugent scoring system exhibits superior sensitivity and specificity compared to the Amsel criteria, (55) its use remains largely restricted to research protocols. Furthermore, questions regarding the risk of potential morbidities and the need for antimicrobial therapy in those women found to have “intermediate flora” remain unanswered. (56, 57)
Several alternative diagnostic methodologies focusing upon the detection of microbial virulence factors produced by the various BV-associated organisms have been proposed in recent years. These include detection of bacterial sialidases, determination of amine levels, and measurement of proline aminopeptidase activity. (58-60) While these techniques are relatively simple, rapid and inexpensive, they fail to identify the specific microbial pathogens present. A potential role for novel, molecular-based techniques for diagnosing BV has recently emerged. Importantly, preliminary studies evaluating these PCR-based strategies have provided additional evidence for G. vaginalis as the primary etiologic agent of BV. (61-63) Menard et al. analyzed 213 vaginal samples from pregnant women using molecular probes targeting 8 BV-related organisms. (64) These authors report that an increased load of G. vaginalis (>109 copies of G. vaginalis DNA per ml) had both high negative and positive predictive values for the diagnosis of BV. While these molecular based diagnostic strategies are promising, the required expertise, laboratory resources and expense limits their use in the primary care setting.
G. vaginalis produces a cholesterol-dependent cytolysin (CDC) (protein pore-forming toxin) called vaginolysin (“VLY”), that acts as a hemolysin (8, 35, 50). IgA-mediated immune responses to the hemolysin occur during BV and are useful as a marker of disease (8, 35). However, complete characterization of the VLY has been limited by the absence of genetic information and an inability to produce recombinant toxin. Therefore there is a great need to sequence and characterize the human-specific VLY toxin, and for methods for treating or preventing G. vaginalis and BV.
The global impact of the HIV epidemic cannot be overstated. Even as antiretroviral drugs prolong and improve life for HIV-infected people in wealthy nations, millions of people suffer and ultimately perish from the ravages of the disease worldwide. Despite this, and despite decades of research, prevention and cure of HIV remain elusive goals. Thus, there is a need for novel and creative approaches to preventing HIV acquisition. BV is exceedingly common, especially in Africa where more than 50% of women in numerous trials, including the recent trial of acyclovir for HSV suppression in Tanzania 1) were infected with BV. BV has been repeatedly associated with both increased risk of HIV acquisition and increased viral shedding among those already infected with HIV2. In vitro, treatment of HIV-infected cells with Gardnerella leads to increased production of viral transcripts. Comparatively little attention has been paid to targeting BV as a means of affecting the progression of the HIV epidemic due to several factors: (1) BV is not a “traditional” sexually transmitted infection (STI) and is often omitted from analyses of STI-HIV interactions; (2) Gardnerella is enigmatic: difficult to culture, without an available genome sequence, lacking known virulence factors, and (perhaps most importantly) without an animal model; (3) BV and Gardnerella colonization are extremely difficult to eradicate even with targeted antibiotic therapy. A randomized, controlled trial of mass antibiotic treatment targeting STI in Uganda did not affect the prevalence of BV in either the control or the treatment group, emphasizing the challenges of BV prevention and treatment (and failing to address the impact of an efficacious BV therapy) (4).
Therefore there is a great need for a new methods and compositions to G. vaginalis and BV to minimize the risk of transmitting HIV from person to person, particularly from an HIV-positive mother who has G. vaginalis to a fetus or an infant during delivery.